Saline mine water influences eukaryote life in shallow groundwater of a tropical sandy stream.

Eukaryotic communities in groundwater may be particularly sensitive to disturbance because they are adapted to stable environmental conditions and often have narrow spatial distributions. Traditional methods for characterising these communities, focussing on groundwater-inhabiting macro- and meiofauna (stygofauna), are challenging because of limited taxonomic knowledge and expertise (particularly in less-explored regions), and the time and expense of morphological identification. The primary objective of this study was to evaluate the vulnerability of eukaryote communities in shallow groundwater to mine water discharge containing elevated concentrations of magnesium (Mg) and sulfate (SO4). The study was undertaken in a shallow sand bed aquifer within a wet-dry tropical setting. The aquifer, featuring a saline mine water gradient primarily composed of elevated Mg and SO4, was sampled from piezometers in the creek channel upstream and downstream of the mine water influence during the dry season when only subsurface water flow was present. Groundwater communities were characterised using both morphological assessments of stygofauna from net samples and environmental DNA (eDNA) targeting the 18S rDNA and COI mtDNA genes. eDNA data revealed significant shifts in community composition in response to mine waters, contrasting with findings from traditional morphological composition data. Changes in communities determined using eDNA data were notably associated with concentrations of SO42-, Mg2+ and Na+, and water levels in the piezometers. This underscores the importance of incorporating molecular approaches in impact assessments, as relying solely on traditional stygofauna sampling methods in similar environments may lead to inaccurate conclusions about the responses of the assemblage to studied impacts.

Shedding light on the Ophel biome: the trans-Tethyan phylogeography of the sulfide shrimp Tethysbaena (Peracarida: Thermosbaenacea) in the Levant.

Background: Tethysbaena are small peracarid crustaceans inhabiting extreme environments such as subterranean lakes and thermal springs, represented by endemic species found around the ancient Tethys, including the Mediterranean, Arabian Sea, Mid-East Atlantic, and the Caribbean Sea. Two Tethysbaena species are known from the Levant: T. relicta, found along the Dead Sea-Jordan Rift Valley, and T. ophelicola, found in the Ayyalon cave complex in the Israeli coastal plain, both belonging to the same species-group based on morphological cladistics. Along the biospeleological research of the Levantine subterranean fauna, three biogeographic hypotheses determining their origins were proposed: (1) Pliocenic transgression, (2) Mid-late Miocenic transgression, and (3) The Ophel Paradigm, according to which these are inhabitants of a chemosynthetic biome as old as the Cambrian. Methods: Tethysbaena specimens of the two Levantine species were collected from subterranean groundwaters. We used the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear ribosomal 28S (28S rRNA) gene to establish the phylogeny of the Levantine Tethysbaena species, and applied a molecular clock approach for inferring their divergence times. Results: Contrary to the morphological cladistic-based classification, we found that T. relicta shares an ancestor with Tethysbaena species from Oman and the Dominican Republic, whereas the circum-Mediterranean species (including T. ophelicola) share another ancestor. The mean age of the node linking T. relicta from the Dead Sea-Jordan Rift Valley and Tethysbaena from Oman was 20.13 MYA. The mean estimate for the divergence of T. ophelicola from the Mediterranean Tethysbaena clade dated to 9.46 MYA. Conclusions: Our results indicate a two-stage colonization of Tethysbaena in the Levant: a late Oligocene transgression, through a marine gulf extending from the Arabian Sea, leading to the colonization of T. relicta in the Dead Sea-Jordan Rift Valley, whereas T. ophelicola, originating from the Mesogean ancestor, inhabited anchialine caves in the coastal plain of Israel during the Mid-Miocene.

Groundwater environmental DNA metabarcoding reveals hidden diversity and reflects land-use and geology.

Despite being the most important source of liquid freshwater on the planet, groundwater is severely threatened by climate change, agriculture, or industrial mining. It is thus extensively monitored for pollutants and declines in quantity. The organisms living in groundwater, however, are rarely the target of surveillance programmes and little is known about the fauna inhabiting underground habitats. The difficulties accessing groundwater, the lack of expertise, and the apparent scarcity of these organisms challenge sampling and prohibit adequate knowledge on groundwater fauna. Environmental DNA (eDNA) metabarcoding provides an approach to overcome these limitations but is largely unexplored. Here, we sampled water in 20 communal spring catchment boxes used for drinking water provisioning in Switzerland, with a high level of replication at both filtration and amplification steps. We sequenced a portion of the COI mitochondrial gene, which resulted in 4917 ASVs, yet only 3% of the reads could be assigned to a species, genus, or family with more than 90% identity. Careful evaluation of the unassigned reads corroborated that these sequences were true COI sequences belonging mostly to diverse eukaryotic groups, not present in the reference databases. Principal component analyses showed a strong correlation of the community composition with the surface land-use (agriculture vs. forest) and geology (fissured rock vs. unconsolidated sediment). While incomplete reference databases limit the assignment of taxa in groundwater eDNA metabarcoding, we showed that taxonomy-free approaches can reveal large hidden diversity and couple it with major land-use drivers, revealing their imprint on chemical and biological properties of groundwater.

Taking eDNA underground: Factors affecting eDNA detection of subterranean fauna in groundwater.

Stygofauna are aquatic fauna that have evolved to live underground. The impacts of anthropogenic climate change, extraction and pollution on groundwater pose major threats to groundwater health, prompting the need for efficient and reliable means to detect and monitor stygofaunal communities. Conventional survey techniques for these species rely on morphological identification and can be biased, labour-intensive and often indeterminate to lower taxonomic levels. By contrast, environmental DNA (eDNA)-based methods have the potential to dramatically improve on existing stygofaunal survey methods in a large range of habitats and for all life stages, reducing the need for the destructive manual collection of often critically endangered species or for specialized taxonomic expertise. We compared eDNA and haul-net samples collected in 2020 and 2021 from 19 groundwater bores and a cave on Barrow Island, northwest Western Australia, and assessed how sampling factors influenced the quality of eDNA detection of stygofauna. The two detection methods were complementary; eDNA metabarcoding was able to detect soft-bodied taxa and fish often missed by nets, but only detected seven of the nine stygofaunal crustacean orders identified from haul-net specimens. Our results also indicated that eDNA metabarcoding could detect 54%-100% of stygofauna from shallow-water samples and 82%-90% from sediment samples. However, there was significant variation in stygofaunal diversity between sample years and sampling types. The findings of this study demonstrate that haul-net sampling has a tendency to underestimate stygofaunal diversity and that eDNA metabarcoding of groundwater can substantially improve the efficiency of stygofaunal surveys.

Subterranean carbon flows from source to stygofauna: a case study on the atyid shrimp Stygiocaris stylifera (Holthuis, 1960) from Barrow Island (WA).

Groundwater biota are crucial for the ecological functioning of subterranean ecosystems. However, while knowledge of the taxonomic diversity of groundwater invertebrates (stygofauna) is increasing, functional ecological information is still limited. Here, we investigate seldom empirically tested assumptions around stygofaunal trophic plasticity in coping with oligotrophic habitats. We focus on Barrow Island (Western Australia), an ideal natural laboratory due to the occurrence of natural oil seeps in association with aquifers. The trophic position and food source use of the endemic atyid shrimp Stygiocaris stylifera (Holthuis, 1960) were assessed via δ13C and δ15N stable isotope analysis (SIA). Background information on the environmental conditions was gathered through hydrochemical data and δ13C SIA combined with 14C data from dissolved inorganic/organic carbon and particulate organic carbon from groundwater samples. Our results indicate carbon enrichment in proximity to the natural oil seepage coupled with changes in trophic positions of S. stylifera from higher consumers/predators to biofilm grazers/decomposers. These results are consistent with an increased involvement of hydrocarbon seeps and associated microbial communities in the carbon flows and confirm potential for the trophic flexibility in stygofauna. Further investigations involving other trophic groups will help elucidate the functioning of the ecosystems at a community level.

Groundwater fauna downtown - Drivers, impacts and implications for subsurface ecosystems in urban areas.

Groundwater fauna (stygofauna) comprises organisms that have adapted to the dark subterranean environment over a course of thousands and millions of years, typically having slow metabolisms and long life cycles. They are crucial players in the groundwater of oxygenic aquifers, and contribute to various ecosystem services. Today's knowledge of their sensitivity to anthropogenic impacts is incomplete and a critical analysis of the general relevance of local findings is lacking. In this review, we focus on those areas with the highest interference between humans and stygofauna: cities. Here is where local pollution by various contaminants and heat strongly stresses the unique groundwater ecosystems. It is demonstrated that it is difficult to discern the influence of individual factors from the findings reported in field studies, and to extrapolate laboratory results to field conditions. The effects of temperature increase and chemical pollution vary strongly between tested species and test conditions. In general, previous findings indicate that heating, especially in the long-term, will increase mortality, and less adapted species are at risk of vanishing from their habitats. The same may be true for salinity caused by road de-icing in cold urban areas. Furthermore, high sensitivities were shown for ammonium, which will probably be even more pronounced with rising temperatures resulting in altered biodiversity patterns. Toxicity of heavy metals, for a variety of invertebrates, increases with time and chronic exposure. Our current knowledge reveals diverse potential impacts on groundwater fauna by urban pollution, but our insights gained so far can only be validated by standardized and long-term test concepts.

eDNA in subterranean ecosystems: Applications, technical aspects, and future prospects.

Monitoring of biota is pivotal for the assessment and conservation of ecosystems. Environments worldwide are being continuously and increasingly exposed to multiple adverse impacts, and the accuracy and reliability of the biomonitoring tools that can be employed shape not only the present, but more importantly, the future of entire habitats. The analysis of environmental DNA (eDNA) metabarcoding data provides a quick, affordable, and reliable molecular approach for biodiversity assessments. However, while extensively employed in aquatic and terrestrial surface environments, eDNA-based studies targeting subterranean ecosystems are still uncommon due to the lack of accessibility and the cryptic nature of these environments and their species. Recent advances in genetic and genomic analyses have established a promising framework for shedding new light on subterranean biodiversity and ecology. To address current knowledge and the future use of eDNA methods in groundwaters and caves, this review explores conceptual and technical aspects of the application and its potential in subterranean systems. We briefly introduce subterranean biota and describe the most used traditional sampling techniques. Next, eDNA characteristics, application, and limitations in the subsurface environment are outlined. Last, we provide suggestions on how to overcome caveats and delineate some of the research avenues that will likely shape this field in the near future. We advocate that eDNA analyses, when carefully conducted and ideally combined with conventional sampling techniques, will substantially increase understanding and enable crucial expansion of subterranean community characterisation. Given the importance of groundwater and cave ecosystems for nature and humans, eDNA can bring to the surface essential insights, such as study of ecosystem assemblages and rare species detection, which are critical for the preservation of life below, as well as above, the ground.

High diversity and local endemism in Aotearoa New Zealand's groundwater crustacean fauna.

We used DNA barcoding to assess the diversity and distribution of New Zealand's groundwater amphipods and isopods (Crustacea) and to determine whether biodiversity and endemism within tectonically active New Zealand are similar to those of more tectonically stable continents. Sixty-five wells were sampled in seven aquifers across four regions within the North and South islands of New Zealand, and resident invertebrates were morphologically identified and then assessed using sequencing of the mitochondrial DNA cytochrome c oxidase subunit one (COI) gene. Invertebrates were found in 54 wells. Of the 228 individual amphipods and isopods found in 36 of the wells, 154 individuals were successfully sequenced for COI (68% success rate) from 25 wells, with at least one well in each aquifer containing sequenced individuals. Of the 45 putative species identified using Barcode Index Numbers (BINs), 30 BINs (78% of all taxa and 83% of amphipods) were previously unrecorded. Substantial morphologically cryptic, species-level diversity was revealed, particularly within the amphipod Family Paraleptamphopidae. Similarly, one isopod taxon morphologically identified as Cruregens fontanus was assigned to five well-separated BINs based on COI sequences. Endemism appeared high, with all taxa regionally endemic; 87% of species were restricted to one aquifer and more than 50% restricted to one well. Non-saturated species accumulation curves indicated that, while additional sampling may increase the range of some currently identified taxa, additional range-restricted taxa are also likely to be discovered. Patterns of diversity and short-range endemism were similar to those found elsewhere, including locations which are more tectonically stable. The predominance of local endemism within New Zealand's groundwater fauna suggests that land-use activities and groundwater extraction require careful evaluation to minimize threats to groundwater biodiversity.

Four scenarios of environmental risk of diclofenac in European groundwater ecosystems.

Groundwater is the largest source of liquid freshwater on Earth. Groundwater ecosystems harbor a rich biodiversity, mainly consisting of microbes and invertebrates that provide substantial ecological services. Despite its importance, groundwater is affected by several anthropic pressures, including pollution from pharmaceutical compounds. Diclofenac is the non-steroidal drug most widely detected in freshwaters, both in surface waters (e.g., rivers, streams, lakes etc.) and groundwaters. Unlike surface waters, the environmental risk of diclofenac in European groundwaters has not yet been assessed by the competent Authorities. The environmental risk assessment refers to the analysis of the potential risk that a chemical compound poses to a given environment by comparing its measured environmental concentrations to its predicted no-effect concentration. In this study, we explored four environmental risk scenarios in European groundwaters using different methodologies. We obtained diverse risk expectations, some indicative of a moderately diffuse environmental risk for concentrations of diclofenac ≥42 ng/L and others indicative of a widespread environmental risk for concentrations ≥5 ng/L. The difference among the four scenarios mainly related to the methods of calculating the predicted no-effect concentration of diclofenac. We discussed the four scenarios in order to identify the most realistic risk expectations posed by diclofenac to European groundwater ecosystems.

Sensitivity of a widespread groundwater copepod to different contaminants.

Groundwater is an indispensable resource for humankind and sustainable biomes functioning. Anthropogenic disturbance threatens groundwater ecosystems globally, but to which extent groundwater organisms respond to stressors remains poorly understood. Groundwater animals are rare, with small populations, difficult to find and to breed in the lab, which poses a main challenge to the assessment of their responses to pollutants. Despite the difficulties, assessing the toxicity of a large spectrum of stressors to groundwater organisms is a priority to inform towards appropriate environmental protection of these ecosystems. We tested the sensitivity to CuSO4, diclofenac, and NaCl of a groundwater population of the copepod Diacyclops crassicaudis crassicaudis and compared its sensitivity with the model organism Daphnia magna. We ranked its sensitivity using a species sensitivity distribution (SSD) approach using the feasible data available for groundwater and surface crustaceans. Our results show that the most toxic compound was CuSO4 for which higher amount of data was recorded and wider variability in response was observed. It was followed by diclofenac, largely lacking data for groundwater-adapted organisms, and the least toxic compound was NaCl. The differential sensitivity between D. crassicaudis and D. magna was contaminant-dependent. As a general trend D. crassicaudis was always distributed in the upper part of the SSD curves together with other groundwater-adapted organisms. Our results highlight that the widespread groundwater populations of the D. crassicaudis species complex, which can be successfully breed in the lab, may provide a reasonable approach to assess the ecological effects of anthropogenic stressors in groundwater ecosystems.

New arietellid copepods (Calanoida, Arietellidae) from anchialine caves in the Eastern Adriatic Sea.

Two new species of calanoid copepods are described; Metacalanus adriaticus sp. nov. from an anchialine cave on Vis Island, and Paramisophria tvrtkovici sp. nov. from Orljak Cave, located in the lower part of River Krka estuary, near the town of Sibenik (Croatia). This is the first report of arietellid copepods found in any anchialine cave along the coast of the Adriatic Sea. In M. adriaticus sp. nov. the antennules are asymmetrical in both sexes (the female left antennule is 18-segmented, right 20-segmented; male left 16-segmented and right 20-segmented); the uniramous fifth legs of the female are 2-segmented; the terminal segment of the fifth leg in both sexes is the longest; and in the male the fifth leg exopod is 2-segmented. In P. tvrtkovici sp. nov. the antennules of both sexes are asymmetrical with the left antennule longer than the right, the female antennule is 21-segmented on both sides; the male left antennule is 19-segmented, the right 21-segmented; the armature of the terminal exopod segment of leg 1 is II, 2 ,2; the male fifth legs have a rudimentary endopod on the left leg, the third exopodal segment is smallest and bears three unequal processes on its outer margin, and the terminal spine is completely separated from the segment. On the right leg the third segment carries two short, unequal processes on its outer distal margin, as well as a long sigmoidal spine which is fused to the segment. It is inferred that, after the last glaciation, these new Arietellids moved out from their southern Adriatic refuge, colonizing first the anchialine habitats of the outer eastern Adriatic islands and then spreading along the coast.

The impact of nitrate on the groundwater assemblages of European unconsolidated aquifers is likely less severe than expected.

In this study, we analyzed the structure of the stygobiotic copepod assemblages of an unconsolidated European aquifer (VO), in southern Italy, that has been subject to persistent nitrate contamination for over 15 years. To this end, we monitored 25 bores where groundwater was contaminated only by nitrate, and no other chemical pollutants were reported as being above detection limits from 2009 to 2014. We monitored these bores three times, namely in autumn 2014 and in spring and autumn 2015. We expected that the chronic exposure to high nitrate concentrations had a significant and evident impact on the stygobiotic copepod assemblages. Unexpectedly, the assemblages were highly diversified. The stygobiotic species richness (SSR) accounted 17 species, a value that exceeded the European mean value (SSR = 12 species). However, the species density was only 0.6 species/km2, lower than the European mean value (= 1.6 species/km2). Moreover, the juvenile copepods were numerically less abundant than the adults and the biomass-abundance model showed signs of alteration of the structure of the copepod assemblages. This study highlighted that (i) nitrates, even at high concentrations, probably have a less severe impact on groundwater assemblages of unconsolidated aquifers than expected and (ii) the analysis of population traits and biomasses can detect signs of alteration of these assemblages that would, otherwise, not be visible from the analysis of the sole species richness and abundances.

Terrestrial Gastropoda from the caves of Presidente Olegário, southeastern Brazil / Gastrópodes terrestres de cavernas da região de Presidente Olegário no sudeste brasileiro

Abstract: Samples of terrestrial gastropods were collected year-round in seven caves in Presidente Olegário municipality, Minas Gerais state, southeastern Brazil, during several expeditions from 2012 to 2014. Twenty-four taxa (plus a single freshwater species), mainly stylommatophorans, were found in the material. The following species are reported for the first time for Minas Gerais state: Alcadia iheringi Wagner, 1910 and Helicina sordida King, 1831 (Helicinidae); Cecilioides consobrina (d'Orbigny, 1841) (Ferussaciidae); Entodina gionensis Morretes, 1940 and Scolodonta interrupta (Suter, 1900) (Scolodontidae); Megalobulimus sanctipauli (Ihering & Pilsbry, 1900) (Strophocheilidae); Drymaeus coarctatus (Pfeiffer, 1845) (Bulimulidae); Habroconus semenlini (Moricand, 1846) (Euconulidae); and Solaropsis aff. rosaria (Pfeiffer, 1849) (Solaropsidae). Furthermore, the species Drymaeus iracema (Simone, 2015) and Drymaeus terreus (Simone, 2015) are synonymized with Drymaeus coarctatus (L. Pfeiffer, 1845).

Resumo: Amostras de gastrópodes terrestres foram coletadas em sete cavernas na região do município de Presidente Olegário, Minas Gerais, Brasil, durane múltiplas expedições ao longo dos anos de 2012 a 2014. Vinte-e-quatro táxons (mais uma única espécie dulciaquícola) foram encontrados, em sua maioria Stylommatophora. As seguintes espécies são aqui reportadas pela primeira vez para o estado de Minas Gerais: Alcadia iheringi Wagner, 1910 e Helicina sordida King, 1831 (Helicinidae); Cecilioides consobrina (d'Orbigny, 1841) (Ferussaciidae); Entodina gionensis Morretes, 1940 e Scolodonta interrupta (Suter, 1900) (Scolodontidae); Megalobulimus sanctipauli (Ihering & Pilsbry, 1900) (Strophocheilidae); Drymaeus coarctatus (Pfeiffer, 1845) (Bulimulidae); Habroconus semenlini (Moricand, 1846) (Euconulidae); e Solaropsis aff. rosaria (Pfeiffer, 1849) (Solaropsidae). Além disso, as espécies Drymaeus iracema (Simone, 2015) e Drymaeus terreus (Simone, 2015) são aqui sinonimizadas com Drymaeus coarctatus (L. Pfeiffer, 1845).

Four new Cyclopina (Copepoda, Cyclopinidae) from South Korea.

Copepods are well studied in South Korea, with the exception of marine non-parasitic cyclopoids, and especially cyclopinids; only three species were found so far here, and only one of them is endemic. A survey of intertidal interstitial faunas from sandy beaches revealed four endemic members of the genus Cyclopina Claus, 1863, which represents the first record of the largest cyclopinid genus in South Korea. A detailed study of their morphology revealed numerous differences, including in rarely studied cuticular organs. Some of these micro-characters could easily be homologised and showed little intraspecific variability, which might prove invaluable for matching sexes and reconstructing phylogenetic relationships. Cyclopina busanensis sp. nov. is described from both sexes collected near Busan (South Coast of South Korea), and is most similar to the only congener from Japan: C. kiraensis Horomi, 1984. Cyclopina koreana sp. nov. is described from both sexes collected near Gangneung (East Coast), and has no close relatives among currently known species. Cyclopina curtijeju sp. nov. is described from two females from Jeju (off South Coast); it is possibly closely related to C. smirnovi Herbst, 1982, but the latter is known from a single male from the Russian Far East. Cyclopina wido sp. nov. is described from both sexes from Wido (West Coast), and shows numerous reductions in segmentation and armature of appendages, most of them probably a consequence of its diminutive size. A table of 26 discrete and continuous characters commonly used in the taxonomy of this group is provided for 48 valid species and subspecies of Cyclopina.

An annotated checklist of the Niphargidae (Crustacea: Amphipoda) of Greece.

Despite Greece being a global hotspot of subterranean biodiversity, its hypogean fauna is largely neglected from both an ecological and conservational point of view. An overview of the Niphargidae occurring in Greece is presented as an annotated list of all available published records. These records have resulted in an updated species list reflecting taxonomic corrections and species distribution range in the Greek peninsula. A total of 23 species, attributed to 3 genera, is up to date known from Greece with a high rate of endemicity found particularly in Crete. The endemic species of Greece amount to 21 (91% of total species richness), with the remaining species distributing also in the Republic of North Macedonia. Currently, none of them is listed in the national, European or global IUCN Red Lists of Threatened Species. Considering the increasing habitat degradation due to anthropic pressure, groundwater harvesting and climate change we could lose rare and endemic species without even acknowledging their existence.

Refining trophic dynamics through multi-factor Bayesian mixing models: A case study of subterranean beetles.

Food web dynamics are vital in shaping the functional ecology of ecosystems. However, trophic ecology is still in its infancy in groundwater ecosystems due to the cryptic nature of these environments. To unravel trophic interactions between subterranean biota, we applied an interdisciplinary Bayesian mixing model design (multi-factor BMM) based on the integration of faunal C and N bulk tissue stable isotope data (δ13C and δ15N) with radiocarbon data (Δ14C), and prior information from metagenomic analyses. We further compared outcomes from multi-factor BMM with a conventional isotope double proxy mixing model (SIA BMM), triple proxy (δ13C, δ15N, and Δ14C, multi-proxy BMM), and double proxy combined with DNA prior information (SIA + DNA BMM) designs. Three species of subterranean beetles (Paroster macrosturtensis, Paroster mesosturtensis, and Paroster microsturtensis) and their main prey items Chiltoniidae amphipods (AM1: Scutachiltonia axfordi and AM2: Yilgarniella sturtensis), cyclopoids and harpacticoids from a calcrete in Western Australia were targeted. Diet estimations from stable isotope only models (SIA BMM) indicated homogeneous patterns with modest preferences for amphipods as prey items. Multi-proxy BMM suggested increased-and species-specific-predatory pressures on amphipods coupled with high rates of scavenging/predation on sister species. SIA + DNA BMM showed marked preferences for amphipods AM1 and AM2, and reduced interspecific scavenging/predation on Paroster species. Multi-factorial BMM revealed the most precise estimations (lower overall SD and very marginal beetles' interspecific interactions), indicating consistent preferences for amphipods AM1 in all the beetles' diets. Incorporation of genetic priors allowed crucial refining of the feeding preferences, while integration of more expensive radiocarbon data as a third proxy (when combined with genetic data) produced more precise outcomes but close dietary reconstruction to that from SIA + DNA BMM. Further multidisciplinary modeling from other groundwater environments will help elucidate the potential behind these designs and bring light to the feeding ecology of one the most vital ecosystems worldwide.

Fundamental research questions in subterranean biology.

Five decades ago, a landmark paper in Science titled The Cave Environment heralded caves as ideal natural experimental laboratories in which to develop and address general questions in geology, ecology, biogeography, and evolutionary biology. Although the 'caves as laboratory' paradigm has since been advocated by subterranean biologists, there are few examples of studies that successfully translated their results into general principles. The contemporary era of big data, modelling tools, and revolutionary advances in genetics and (meta)genomics provides an opportunity to revisit unresolved questions and challenges, as well as examine promising new avenues of research in subterranean biology. Accordingly, we have developed a roadmap to guide future research endeavours in subterranean biology by adapting a well-established methodology of 'horizon scanning' to identify the highest priority research questions across six subject areas. Based on the expert opinion of 30 scientists from around the globe with complementary expertise and of different academic ages, we assembled an initial list of 258 fundamental questions concentrating on macroecology and microbial ecology, adaptation, evolution, and conservation. Subsequently, through online surveys, 130 subterranean biologists with various backgrounds assisted us in reducing our list to 50 top-priority questions. These research questions are broad in scope and ready to be addressed in the next decade. We believe this exercise will stimulate research towards a deeper understanding of subterranean biology and foster hypothesis-driven studies likely to resonate broadly from the traditional boundaries of this field.

Do Chinese cavefish show intraspecific variability in morphological traits?

Cavefishes represent one of the most bizarre and intriguing life forms inhabiting groundwater environments. One-third of the known cavefishes worldwide is endemic to China, and almost half of those belongs to a single genus, Sinocyclocheilus (Cypriniformes: Cyprinidae). Analyzing the morphometrics of three Sinocyclocheilus species, we aimed to assess whether variability among conspecific populations exists. We predict that populations inhabiting different subterranean habitats (shallow vs. deep) show divergences in specific morphological traits to better cope with the local ecological conditions. Our results showed that the populations showing bigger eyes and reduced humpback were those occurring close to the cave entrance (habitats with light and high food availability), while specimens with smaller eyes and increased humpback were collected from deeper groundwater areas (habitats laying in darkness with food scarcity). This explorative study paves the way for further researches aiming to collect novel data on Chinese cavefishes and highlights the usefulness of these species in evolutionary studies.

Ecotoxicological effects of anthropogenic stressors in subterranean organisms: A review.

How anthropogenic stressors affect biodiversity is a central question in a changing world. Subterranean ecosystems and their biodiversity are particularly vulnerable to change, yet, these species are frequently neglected in analyses of global biodiversity and assessments of ecological status and risk. Are these hidden species affected by anthropogenic stressors? Do they survive outside of the current thermal limits of their ecosystems? These and other important questions can be addressed with ecotoxicological testing, relating contaminants and temperature resistance of species with measured environmental concentrations and climatic data. Ecotoxicological knowledge specific to subterranean ecosystems is crucial for establishing thresholds for their protection, but such data are both scarce and scattered. Here, we review the existing ecotoxicological studies of these impacts to subterranean-adapted species. An effort that includes 167 measured endpoints and presents a database containing experimentally derived species' tolerance data for 28 contaminants and temperature, for 46 terrestrial and groundwater species, including fungi and animals. The lack of standard data among the studies is currently the major impediment to evaluate how stressors affect subterranean-adapted species and how differently they respond from their relatives at surface. Improving understanding of ecotoxicological effects on subterranean-adapted species will require extensive analysis of physiological responses to a wide range of untested stressors, standardization of testing protocols and evaluation of exposures under realistic scenarios.

A new species of stygobitic snail in the genus Antrorbis Hershler & Thompson, 1990 (Gastropoda, Cochliopidae) from the Appalachian Valley and Ridge of eastern Tennessee, USA.

A new species of cave snail (Littorinimorpha: Cochliopidae) in the genus Antrorbis is described from the dark zone of two caves in the Appalachian Valley and Ridge province in eastern Tennessee, United States. The Tennessee Cavesnail, Antrorbis tennesseensis Perez, Shoobs, Gladstone, & Niemiller, sp. nov. is distinguished from its only known congener, Antrorbis breweri, by the absence of raised tubercles on its finely spirally striate protoconch, and its unique radular formula. Moreover, A. tennesseensis is genetically distinct from A. breweri based on substantial divergence at the mitochondrial CO1 locus. This is the first cavesnail to be described from the Appalachian Valley and Ridge (AVR) physiographic province in the state of Tennessee, which previously represented a substantial gap in the distribution of stygobitic (i.e., aquatic, subterranean-obligate) gastropods.